Project Summary Each organelle of the cell has a unique lipid and protein composition that confers its specific functions. While much has been learned regarding the mechanisms that mediate sorting of proteins to different organelles, the mechanisms that govern the lipid content of organelles are largely unknown. New lipid synthesis occurs in the endoplasmic reticulum (ER), the Golgi apparatus, and the mitochondrion, and they are then distributed to other organelles. There are expansive gaps in knowledge regarding the mechanisms that coordinate the synthesis and distributions of lipids. The research proposed in this project focuses on sphingomyelin, a major structural component of the cellular membranes, and the Golgi apparatus, which functions as a ?lipid based sorting station? that disseminates lipids and proteins to nearly all organelles of the cell. Multiple types of cargo transport vesicles bud from the Golgi and it is widely thought that coalescence of lipids such as sphingolipids and cholesterol is a driving force for sorting of cargos into to transport pathways. Preliminary data document the engineering of a natural sphingomyelin-binding protein into a biosensor that enables analyses of sphingomyelin dynamics (synthesis, trafficking) in living cells. We will apply this new tool in fluorescence microscopy-based experiments to determine if sphingomyelin is enriched in distinct Golgi-to-plasma membrane transport carriers. Protein ?clients? of the sphingomyelin transport pathway will be identified using enzymatic proximity labeling and proteomic analyses, and sphingomyelin-dependent sorting mechanisms for these proteins will be elucidated. The research will provide new insights into the regulation and roles of sphingomyelin within the cell.